Not Applicable
1. Technical Field
The invention is in the field of analysis of cell populations and cell separation and the compositions obtained thereby. More particularly, the invention concerns analysis and separation of antigen-specific T cells based on primary labeling of cells with their secreted products through capture of these products by a specific binding partner for the product anchored or bound to the cell surface.
2. Background Art
Numerous attempts have been made to analyze populations of cells and to separate cells based on the products which they produce. Such approaches to cell analysis and separation are especially useful in assessing those cells which are capable of secreting a desired product (the xe2x80x9cproductxe2x80x9d), or which are relatively high secretors of the product. These methods include cloning in microtiter plates and analysis of the culture supernatant for product, cloning in agar and analysis by methods for identification of the product of the localized cells; the identification methods include, for example, plaque assays and western blotting. Most methods for analysis and selection of cells based upon product secretion involve physically isolating the cell, followed by incubation under conditions that allow product secretion, and screening of the cell locations to detect the cell or cell clones that produce the product. When cells are in suspension, after the cells have secreted the product, the product diffuses from the cell without leaving a marker to allow identification of the cell from which it was secreted. Thus, secretor cells cannot be separated from non-secretor cells with these types of systems.
In other cases, both secretor and non-secretor cells can associate the product with the cell membrane. An example of this type of system are B cell derived cell lines producing monoclonal antibodies. It has been reported that these types of cell lines were separated by fluorescence activated cell sorting (FACS) and other methods reliant upon the presence of antibody cell surface markers. However, procedures that analyze and separate cells by markers that are naturally associated with the cell surface can not accurately identify and/or be used in the separation of secretor cells from non-secretor cells. In addition, systems such as these are not useful in identifying quantitative differences in secretor cells (i.e., low level secretors from high level secretors).
A method that has been used to overcome the problems associated with product diffusion from the cells has been to place the cell in a medium that inhibits the rate of diffusion from the cell. A typical method has been to immobilize the cell in a gel-like medium (agar), and then to screen the agar plates for product production using a system reliant upon blotting, for example Western blots. These systems are cumbersome and expensive if large numbers of cells are to be analyzed for properties of secretion, non-secretion, or amount of secretion.
Kxc3x6hler et al. have described a negative-selection system in which mutants of a hybridoma line secreting IgM with anti-trinitrophenyl (anti-TNP) specificity were enriched by coupling the hapten (i.e., TNP) to the cell surface and incubating the cells in the presence of complement. In this way, cells secreting wild-type Ig were lysed, whereas cells secreting IgM with reduced lytic activity or not binding to TNP preferentially survived. Kxc3x6hler and Schulman (1980) Eur. J. Immunol. 10:467-476.
More recently, a system has been described for labeling and separating cells based on secreted product. PCT/US93/10126. In this system, a specific binding partner for a secreted product is coupled to the surface of cells. The product is secreted, released, and bound to the cell by the specific binding partner. Cells are then separated based on the degree to which they are labeled with the bound product.
Other systems allow the cells to secrete their products in the context of microdroplets of agarose gel which contain reagents that bind the secretion products, and encapsulation of the cells. Such methods have been disclosed in publications by Nir et al. (1990) Applied and Environ. Microbiol. 56:2870-2875; and Nir et al. (1991) Applied and Environ. Microbiol. 56:3861-3866. These methods are unsatisfactory for a variety of reasons. In the process of microencapsulation, statistical trapping of numbers of cells in the capsules occurs, resulting in either a high number of empty capsules when encapsulation occurs at low cell concentrations, or multiple cells per capsule when encapsulation occurs at high cell concentrations. Secreted product is trapped in the agarose drop by the capture antibody and detected by a second fluorochromated antibody. This process, while allowing for the detection and isolation of cells based on secreted product, is complicated, requires special equipment, and is not suited to all types of sorting methods. In order to analyze and separate single cells or single cell clusters by this technique, large volumes must be handled to work with relatively small numbers of cells because of the numbers of empty capsules and because of the size of the microcapsules (50-100 xcexcm). The large volume of droplets results in background problems using flow cytometry analysis and separation. In addition, the capsules do not allow separation using magnetic beads or panning for cell separation.
Various methods have been used to couple labels to cell surfaces where the label such as a fluorochrome is intended for direct detection. For example, hydrophobic linkers inserted into the cell membrane to couple fluorescent labels to cells have been described in PCT WO 90/02334, published Mar. 8, 1990. Antibodies directed to HLA have also been used to bind labels to cell surfaces. Such binding results in a smaller dimension than the encapsulated droplets described above and such cells can be conveniently used in standard separation procedures including flow cytometry and magnetic separations.
ELISpot assays and methods for intracellular cytokine staining have been used for enumeration and characterization of antigen-specific CD4+ and CD8+ T cells. Lalvani et al. (1997) J. Exp. Med. 186:859-865; and Waldrop et al. (1997) J. Clin Invest. 99:1739-1750. These methods can be quite useful for T-cell epitope mapping or for monitoring immunogenicity in vaccine trials, but they do not allow isolation of live antigen-specific T cells, e.g., for clinical trials of specific adoptive immunotherapy of cancer or infections. Kern et al. (1998) Nat. Med. 4:975-978; E1 Ghazali et al. (1993) Curr. Opin Immunol. 23:2740-2745; and Yee et al (1997) Curr. Opin. Immund. 9:702-708. Soluble multivalent complexes of peptide-loaded major histocompatibility complex (MHC) molecules have been exploited recently to detect and also isolate antigen-specific T cells. Altman et al. (1996) Science 274:94-96; Dunbar et al. (1998) Curr. Biol. 8:413-416; Ogg et al. (1998) 279:2103-2106; Luxembourg et al. (1998) Nat. Biotechnol. 16:281-285; Murali-Krishna et al. (1998) Immunity 8:177-187; Gallimore et al. (1998) J. Exp. Med. 187:1383-1393; and Flynn et al. (1998) Immunity 8:683-691. These reagents are highly specific but the approach is limited to well defined combinations of antigenic peptides and restricting HLA alleles.
The immune system comprises two types of antigen-specific cells: B cells and T cells. T cells can be characterized phenotypically by the manner in which they recognize antigen, by their cell surface markers, and by their secreted products. Unlike B cells, which recognize soluble antigen, T cells recognize antigen only when the antigen is presented to them in the form of small fragments bound to major histocompatibility complex (MHC) molecules on the surface of another cell. Any cell expressing MHC molecules associated with antigen fragments on its surface can be regarded as an antigen-presenting cell (APC). In most situations, however, more than the mere display of an MHC-bound antigen fragment on a cell surface is required to activate a T lymphocyte. In addition to the signal delivered via the T cell receptor (TCR) engaged by MHC molecule plus antigen, the T cell must also receive co-stimulatory signals from the APC. Typically APCs are dendritic cells, macrophages or activated B lymphocytes.
T cells express distinctive membrane molecules. Included among these are the T cell antigen receptor (TCR), which appears on the cell surface in association with CD3; and accessory molecules such as CD5, CD28 and CD45R. Subpopulations of T cells can be distinguished by the presence of additional membrane molecules. Thus, for example, T cells that express CD4 recognize antigen associated with class II MHC molecules and generally function as helper cells, while T cells that express CD8 recognize antigen associated with class I MHC molecules and generally function as cytotoxic cells. The CD4+ subpopulation of T cells can be categorized fturther into at least two subsets on the basis of the types of cytokines secreted by the cell. Thus, while both subsets secrete IL-3 and GM-CSF, TH1 cells generally secrete IL-2, IFN-xcex3, and TNF-xcex1, whereas TH2 cells generally secrete IL-4, IL-5, IL-10, and IL-13.
Minor changes in the peptide bound to the MHC molecule can not affect the affinity of the peptide-MHC molecule interaction, yet they can generate partial signals that lead to a halfway response characterized by proliferation and secretion of only a fraction of the cytokines produced during a full T cell response. Some modified peptides can even block proliferation and cytokine secretion altogether and induce a state of T cell anergy or unresponsiveness. There are thus three different types of peptides: agonist (those that stimulate a full response), partial agonist (those that stimulate a partial response) and antagonist (those that induce unresponsiveness). When a single APC presents a mixture of an agonist and an antagonist on its surface, the negative effect of the latter can overcome the positive effect of the former, even if the antagonist is present in much smaller amounts than the agonist. Some viruses seem to use mutations in their proteins to produce antagonist peptides capable of suppressing the activity of the T cell clones that recognize agonist peptides derived from the original wild-type virus.
Secretion by a T cell of a particular cytokine is generally associated with a particular function. For example, differences in the cytokines secreted by the TH1 and TH2 subsets of CD4+ T cells are believed to reflect different biological functions of these two subsets. The TH1 subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T cells, whereas the TH2 subset functions more effectively as a helper for B-cell activation. The TH1 subset can be particularly suited to respond to viral infections and intracellular pathogens because it secretes IL-2 and IFN-xcex3, which activate cytotoxic T cells. The TH2 subset can be more suited to respond to extracellular bacteria and helminthic parasites and can mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. There is also considerable evidence suggesting that preferential activation of TH1 cells plays a central role in the pathogenesis of a number of autoimmune diseases. Secretion of IL-10 by TH2 cells is thought to suppress, in an indirect manner, cytokine production by TH1 cells, and, accordingly, has a general immunosuppressive effect. A shift in the TH1/TH2 balance can result in an allergic response, for example, or, in an increased cytotoxic T cell response.
The changes initiated by the TCR in the first few minutes to hours of activation lead to transition of the cell from the G0 to G1 phase of the cell cycle. Several hours after stimulation of the T cell begins to express IL-2 and high-affinity IL-2 receptor. IL2 gene expression is effected by a set of transcription factors that are activated by the converging signaling pathways triggered by the ligation of TCR, CD28 and possibly other T cell surface molecules.
The transcription factors also induce expression of the CD25 gene, which encodes the xcex1-subunit of the high-affinity IL-2 receptor. The interaction of IL-2 with the high-affinity receptor initiates signaling pathways that cause the T cell to transit from the G1 to the S phase of the cell cycle and progress to cell division. The signaling pathways control the expression and activity of several key proteins necessary for cell division. Some of these are also activated directly by TCR- and CD28-dependent signals while others are energized only by signals provided via the IL-2 receptor.
The stimulated T cell undergoes a sequence of phenotypic changes beginning with its progression from the resting state to mitosis and later to differentiation into effector and memory cells. Among the earliest (immediate) changes, observable within 15-30 minutes of stimulation, are the expression of genes encoding transcription factors such as c-Fos, NF-AT, c-Myc and NF-xcexaB, protein kinases such as Jak-3 and protein phosphatases such as Pac-1. The subsequent early changes, occurring within several hours of stimulation, mark the beginning of the expression of genes encoding activation antigens. These include several cytokines (IL-2 and others), IL-2 receptor subunit xcex1 (CD25), insulin receptor, transferrin receptor and several other surface molecules such as CD 26, CD30, CD54, CD69 and CD70.
Activation antigens reach a maximum level of expression just before the first division, 24 hours after stimulation. During this period the level of expression of several other molecules already expressed on resting T cells increases. At a later point, some days after activation commenced, late activation antigens become expressed on the T cells. These include MHC class II molecules and several members of the xcex21 integrin family. Expression of late activation antigens marks the differentiation of the activated cell into effector or memory T cells.
T cells play important roles in autoimmunity, inflamation, cytotoxicity, graft rejection, allergy, delayed-type hypersensitivity, IgE-mediated hypersensitivity, and modulation of the humoral response. Disease states can result from the activation of self-reactive T cells, from the activation of T cells that provoke allergic reactions, or from the activation of autoreactive T cells following certain bacterial and parasitic infections, which can produce antigens that mimic human protein, rendering these protein xe2x80x9cautoantigens.xe2x80x9d These diseases include, for example, the autoimmune diseases, autoimmune disorders that occur as a secondary event to infection with certain bacteria or parasites, T cell-mediated allergies, and certain skin diseases such as psoriasis and vasculitis. Furthermore, undesired rejection of a foreign antigen can result in graft rejection or even infertility, and such rejection can be due to activation of specific T lymphocyte populations. Pathological conditions can also arise from an inadequate T cell response to a tumor or a viral infection. In these cases, it would be desirable to increase an antigen-specific T cell response in order to reduce or eliminate the tumor or to eradicate an infection.
Autoimmune diseases have a variety of causes. For instance, autoimmune reactions can be provoked by injury or immunization with collagen, by superantigens, by genetic factors, or errors in immune regulation. Superantigens are polyclonal activators that can, among other things, stimulate clones previously anergized by an encounter with an autoantigen or clones that ignored the potential autoantigens because of their low expression or availability. Certain autoimmune disease are caused mainly by autoantibodies, others are T cell-mediated. Autoreactive T cells cause tissue damage in a number of autoimmune diseases including rheumatoid arthritis and multiple sclerosis.
In the treatment of autoimmune disorders, nonspecific immune suppressive agents have been used to slow the disease; these therapies often cause a general immunosuppression by randomly killing or inhibiting immunocompetent cells. Attempts to treat autoimmune disorders by modulating the activity of autoreactive T cells have included immunization with TCR peptides, treatment with interferon-xcex2 (IFN-xcex2) and T lymphocyte vaccination. Ebers (1994) Lancet 343:275-278; Hohlfeld (1997) Brain 120:865-916; and Hafler et al. (1992) Clin. Immunol. Immunopathol. 62:307-313.
The development of allergic sensitization, contact sensitivity and inflammation is dependent on activation and stimulation of T cells that exhibit pro-allergic functions. Allergen-specific T cells are believed to play an important role in the pathophysiology of atopic allergies. Elimination or suppression of allergen-specific T cells could help ameliorate allergic diseases caused by such T cells.
In the initial phase of an allergic reaction, antigen (allergen) enters the body, is picked up by APCs, displayed by them in the context of class II MHC molecules and recognized by helper T cell precursors. These are stimulated to proliferate and differentiate mainly into TH2 cells, which help B lymphocytes differentiate into antibody-producing plasma cells. As in any other antibody-mediated response, the B cells that receive specific help from TH cells are those that recognized the allergen via their surface receptors. Some of the cytokines produced by the TH2 cells, especially IL-4 and IL-13, stimulate the B cells to effect an immunoglobulin isotype switch and to produce IgE antibodies. The antibodies bind to high-affinity Fc receptors on the surface of mast cells in the connective tissue and mucosa, as well as to those on the surface of basophils in the circulation and mucosa and initiate the manifestations of allergic reaction.
Allograft rejection is caused principally by a cell-mediated immune response to alloantigens (primarily MHC molecules) expressed on cells of the graft. Analysis of the T lymphocyte subpopulations involved in allograft rejection has implicated both CD4+ and CD8+ populations. TH1 cells initiate the inflammatory reaction of delayed-type hypersensitivity, leading to the recruitment of monocytes and macrophages into the graft. Natural kill (NK) cells, presumably alerted by the absence in the graft of MHC molecules present in the recipient, can also attack the graft in the early phases of the response. Neutrophils are mainly responsible for clearing the wound or removing damaged cells and cellular debris in the late phase of the allograft reaction.
Most immunosuppressive treatments developed have the disadvantage of being non-specific; that is, they result in generalized immunosuppression, which places the recipient at increased risk for infection. Immunosuppressive agents employed to prevent organ rejection include mitotic inhibitors such as azathioprine, cyclophosphamide and methotrexate; corticosteroids; and drugs, such as cyclosporin, FK506 and rapamycin, which inhibit the transcription of the genes encoding IL-2 and the high-affinity receptor for IL-2.
In the treatment of cancers, cellular immunotherapy has been employed as an alternative, or an adjunct to, conventional therapies such as chemotherapy and radiation therapy. For example, cytotoxic T lymphocyte (CTL) responses can be directed against antigens specifically or preferentially presented by tumor cells. Following activation with T cell cytokines in the presence of appropriately presented tumor antigen, tumor infiltrating lymphocytes (TILs) proliferate in culture and acquire potent anti-tumor cytolytic properties. Weidmann et al. (1994) Cancer Immunol. Immunother. 39:1-14.
The introduction into a cancer patient of in vitro activated lymphocyte populations has yielded some success. Adoptive immunotherapy, the infusion of immunologically active cells into a cancer patient in order to effect tumor regression, has been an attractive approach to cancer therapy for several decades. Two general approaches have been pursued. In the first, donor cells are collected that are either naturally reactive against the host""s tumor, based on differences in the expression of histocompatibility antigens, or made to be reactive using a variety of xe2x80x9cimmunizingxe2x80x9d techniques. These activated donor cells are then transfused to a tumor-bearing host. In the second general approach, lymphocytes from a cancer patient are collected, activated ex vivo against the tumor and then reinfused into the patient. Triozzi (1993) Stem Cells 11:204-211; and Sussman et al. (1994) Annals Surg Oncol. 1:296.
Current methods of cancer treatment are relatively non-selective. Surgery removes the diseased tissue, radiotherapy shrinks solid tumors and chemotherapy kills rapidly dividing cells. Systemic delivery of chemotherapeutic agents, in particular, results in numerous side effects, in some cases severe enough to preclude the use of potentially effective drugs.
Viral diseases are also candidates for immunotherapy. Heslop et al. (1996) Nature Med. 2:551-555. Immunological responses to viral pathogens are sometimes ineffective in eradicating or sufficiently depleting the virus. Furthermore, the highly mutable nature of certain viruses, such as human immunodeficiency virus, allows them to evade the immune system.
Clearly, there is a need to identify, analyze and enrich populations of T cells involved in the above-mentioned pathologies. Currently, several methods for analysis and for enrichment of antigen-specific and/or cytokine-secreting T cells exist. Enrichment of antigen-specific T cells can be achieved using selective culturing techniques to obtain T cell lines and T cell clones. These techniques generally involve culturing the T cells in vitro over a period of several weeks and using rather cumbersome methods to select lines or clones exhibiting the desired phenotype, such as cytokine secretion. Other attempts to detect and enrich for antigen-specific T cells have employed defined multimeric MHC-antigen and MHC-peptide complexes. U.S. Pat. No. 5,635,363. For such a technique to be successful, however, MHC-antigen complexes of the correct MHC allotype are required, and the selection is limited to antigen specificity, i.e., no selection for cytokine secretion is afforded by this technique.
Intracellular cytokine staining after antigen activation, followed by FACS analysis, is the method used to obtain information regarding the antigen specificity and kinetics of cytokine production. Waldrop et al. (1997) J. Clin. Invest. 99:1739-1750. This method is useful for analysis only, since the cells are not viable after this procedure. Similarly, cytokine ELISPOT assays are useful for analysis only. Miyahira et al. (1995) J. Immunol. Met. 181:45-54; and Lalvani et al. (1997) J. Exp. Med. 186:859-865. In these assays, secreted cytokines are trapped in a surrounding matrix for analysis, but there is no mechanism for identifying and retrieving the cell which secreted the cytokine. The gel microdrop technology is not suited to processing large numbers of cells such as would be necessary for treatment of the above-mentioned indications.
It is apparent from the foregoing discussion that there is a need for reliable techniques for analyzing and separating populations of T cells, based on secreted product, for a number of therapeutic and diagnostic purposes. The present invention addresses this need by providing methods for analyzing, separating and enriching populations of antigen-specific T cells.
The invention provides a method for convenient analysis and cell separation of antigen-specific T cells based on one or more products secreted by these cells in response to antigen stimulation. The T cells are provided with a capture moiety specific for the product (or, xe2x80x9cspecific binding partnerxe2x80x9d), which can then be used directly as a label. The binding of the product to the capture moiety results in a xe2x80x9ccaptured product.xe2x80x9d Alternatively, the cells are bound to the product via the capture moiety and can be further labeled via label moieties that bind specifically to the product and that are, in turn, labeled either directly or indirectly with traditional labeling materials such as fluorophores, radioactive isotopes, chromophores or magnetic particles.
The labeled cells can then be separated using standard cell sorting techniques based on these labels. Such techniques include, but are not limited to, flow cytometry, FACS, high gradient magnetic gradient separation, centrifugation.
Thus, in one aspect, the invention encompasses a method to stimulate and separate antigen-specific T cells from a population of cells according to a product secreted and released by the antigen specific T cells in response to the stimulation. The method comprises stimulating a mixture of cells containing T cells with antigen, and effecting a separation of antigen-stimulated cells according to the degree to which they are labeled with the product. Antigen stimulation is achieved by exposing the cells to at least one antigen under conditions effective to elicit antigen-specific stimulation of at least one T cell. Labeling with the product is achieved by modifying the surface of the cells to contain at least one capture moiety, culturing the cells under conditions in which the product is secreted, released and specifically bound (xe2x80x9ccapturedxe2x80x9d or xe2x80x9centrappedxe2x80x9d) to said capture moiety; and labeling the captured product with a label moiety, where the labeled cells are not lysed as part of the labeling procedure or as part of the separation procedure.
Another aspect of the invention is a composition of matter containing antigen-specific T cells capable of capturing a product secreted and released by these cells in response to antigen stimulation, where the surface of the cells is modified to contain a capture moiety for the product. The captured product can be separately labeled by a label moiety.
Still another aspect of the invention is antigen-specific T cells and progeny thereof separated by the above-described method.
Yet another aspect of the invention is a method to label antigen-specific T cells with a product secreted and released by the cells in response to antigen stimulation, by modifying the surface of these cells to contain a specific binding partner for the product coupled to the cell surface, and culturing the cells under conditions wherein the product is secreted and released.
An additional aspect of the invention is a method of analyzing a population of antigen-specific T cells to determine the proportion of cells that secrete an amount of product relative to other cells in the population, where the product is secreted in response to antigen stimulation. The method comprises labeling the cells by the above-described method, further labeling the cells with a second label that does not label the captured product, and detecting the amount of product label relative to the second cell label. Such methods are useful, for example, in assessing the immune status of an individual.
A further aspect of the invention is methods for use of T cell populations enriched in antigen-specific T cells. The methods comprise administering to an individual in need of treatment a composition comprising a T cell population enriched in antigen-specific T cells. Such methods are useful to treat a variety of pathological conditions, including cancer, allergies, immunodeficiencies, autoimmune disorders, and viral diseases.
Yet another aspect of the invention is a kit for use in separation of antigen-specific T cells from a mixed population comprising effector cells. The kit can contain a physiologically acceptable medium which can be of varying degrees of viscosity up to a gel-like consistency, a product capture system comprising anchor and capture moieties; a label system for detecting the captured product; and instructions for use of the reagents, all packaged in appropriate containers. Optionally, the kit further comprises a magnetic labeling system and/or one or more biological modifiers.
Still another aspect of the invention is a kit for use in the detection/separation of antigen-specific T cells that secrete a desired product in response to antigen stimulation, the kit comprising a product capture system comprising anchor and capture moieties; a label system for detecting the captured product; and instructions for use of the reagents, all packaged in appropriate containers. Optionally, the kit further comprises a magnetic labeling system, and/or antigen, and/or one or more biological modifiers.